The maintenance of normal CNS functions in the aging brain requires constant supply of de novo astrocytes that originate from neural stem cells/progenitors present in the adult brain. Astrocytes of the adult subventricular zone can function as stem cells and differentiate into neurons. How these processes are regulated is currently not well known. Identification of factors that control embryonic neurogliagenesis and differentiation could help to understand the regulation of the adult process. The nuclear protein g-sept is expressed during embryonic neurogliagenesis and differentiation (Dobi et al, 2000). Blocking g-sept binding to its DNA site in embryonic neuroglia cultures results in a decreased number of GFAP+ and an increased number of nestin+ proliferating (BrdU+) cells. We have purified the g-sept protein using DNA-affinity chromatography. Sequence analysis showed that g-sept share homology with members of the heterogeneous nuclear ribonucleoprotein A (hnRNP A) family. The hypothesis of this proposal is that g-sept regulates neurogliagenesis and/or differentiation. The objective of this proposal is to characterize g-sept at the molecular level and to determine its functional significance in neurogliagenesis / differentiation. The Specific Aims are: 1. Molecular characterization of g-sept: full length g-sept cDNA will be obtained by PCR using degenerate primers from an embryonic rat forebrain cDNA library and by 5' and 3' RACE; the primary structure of g-sept cDNA will be determined and its relationship to known proteins will be determined; g-sept will be expressed and the recombinant protein will be characterized by Southwestern analysis and by EMSA. These experiments will provide information about the primary structure of g-sept and also reagents for functional studies. 2. Determine the functional significance of g-sept in neurogliagenesis. The availability of g-sept will be altered by antisense treatment and/or by overexpression of the protein in embryonic neuroglia cultures; at various time points following treatments, cultures will be analyzed for nestin and GFAP expression and for proliferation by immunohistochemistry and by BrdU incorporation; the pattern of g-sept expression in the embryonic (E18) and in the adult rat brain will determined by in situ hybridization histochemistry. These studies will provide basic information about the expression and about the role of g-sept in neurogliagenesis. Results will be used to design detailed studies focusing on the regulation of neurogliagenesis in the aging brain.